Development of Protocol for Production of Primary Antibody against Ovalbumin Protein in Chicken for Detection of the Protein through Western Blotting

Ovalbumin, a major protein of egg white plays many roles including providing nutrition to the developing embryo, acting as coagulating agent, folliculogenesis and angiogenesis in chicken and other animals. This protein is expressed mainly in magnum and then deposited over the yolk of the oocyte/zygote. Hence, it is important in formation of egg and is an essential target to measure. We cloned chicken ovalbumin CDS in pAcGFP-C1 vector and has been initially expressed in chicken primary magnum cell culture. The ovalbumin protein tagged with 6x Histidine was purified from cell culture and used for production of primary antibody in rat. The ovalbumin protein along with freund’s adjuvant was injected to the rat, booster was given, and finally, hyper-immune sera was collected from rat. The antisera was purified for isolation of IgG. The IgG was used as primary antibody for Western blotting. Through Western blotting, ovalbumin protein isolated from chicken magnum was detected and the protocol was established to detect chicken ovalbumin protein.

Hepatitis B prevalence during pregnancy.

In order to determine the efficacy of a new hepatitis B immune globulin (HBIG), a phase 3, vertical transmission (mother to child) clinical interventional trial of hepatitis B virus (HBV) post exposure prophylaxis (PEP) was conducted at selected sites (n=15) throughout India. This required a large screening program for HBsAg positivity at prenatal clinics located in tertiary care hospitals. 36,379 pregnant women consented to be tested for Hepatitis B surface antigen (HBsAg) by Rapid Test and if positive-confirmed by ELISA. The weighted mean prevalence was 0.82% (95% CI, 0.72, 0.91). In conclusion, the prevalence of HBV carrier state during pregnancy in India in this study was low compared to previous reports.

Antifilarial activity of Caesalpinia bonducella against experimental filarial infections.

BACKGROUND & OBJECTIVE: Lymphatic filariasis is a disabling disease that continues to cripple population in tropical countries. Currently available antifilarial drugs are not able to control the disease. Therefore, a better antifilarial is urgently required for proper management of the disease. We undertook this study to assess the antifilarial activity of Caesalpinia bonducella-seed kernel against rodent filarial parasite in experimental model. METHODS: Microfilaraemic cotton rats and Mastomys coucha harbouring Litomosoides sigmodontis and Brugia malayi respectively, were treated with crude extract or fractions of the seed kernel C. bonducella through oral route for 5 consecutive days. Microfilaricidal, macrofilaricidal and female worm sterilizing efficacy was assessed. RESULTS: Crude extract showed gradual fall in microfilariae (mf) count in L. sigmodontis-cotton rat model from day 8 post-treatment attaining more than 95 per cent fall by the end of observation period. It also exhibited 96 per cent macrofilaricidal and 100 per cent female sterilizing efficacy. The butanol fraction F018 caused 73.7 per cent reduction in mf count and 82.5 per cent mortality in adult worms with 100 per cent female sterilization. The aqueous fraction F019 exerted more than 90 per cent microfilaricidal activity and 100 per cent worm sterilization. Two chromatographic fractions, F024 and F025 of hexane soluble fraction exhibited 64 and 95 per cent macrofilaricidal activity, respectively. Both the fractions caused gradual fall in microfilaraemia and 100 per cent worm sterilization. In B. malayi-M. coucha model F025 showed gradual reduction in microfilaraemia and caused 80 per cent sterilization of female parasites INTERPRETATION & CONCLUSION: In conclusion, C. bonducella- seed kernel extract and fractions showed microfilaricidal, macrofilaricidal and female-sterilizing efficacy against L. sigmodontis and microfilaricidal and female-sterilizing efficacy against B. malayi in animal models, indicating the potential of this plant in providing a lead for new antifilarial drug development.

Drosophila simulans Lethal hybrid rescue mutation (Lhr) rescues inviable hybrids by restoring X chromosomal dosage compensation and causes fluctuating asymmetry of development.

The Drosophila simulans Lhr rescues lethal hybrids from the cross of D. melanogaster and D. simulans. We describe here, the phenotypes of Lhr dependent rescue hybrids and demonstrate the effects of Lhr on functional morphology of the salivary chromosomes in the hybrids. Our results reveal that the phenotypes of the 'Lhr dependent rescued' hybrids were largely dependent on the genetic background and the dominance in species and hybrids, and not on Lhr. Cytological examination reveal that while the salivary chromosome of 'larval lethal' male carrying melanogaster X chromosome was unusually thin and contracted, in 'rescued' hybrid males (C(mel)X(mel)Y(sim); A(mel)A(sim)) the X chromosome showed typical pale staining, enlarged diameter and incorporated higher rate of (3)H-uridine in presence of one dose Lhr in the genome. In hybrid males carrying simulans X chromosome (C(mel)X(sim)Y(mel); A(mel)A(sim)), enlarged width of the polytene X chromosome was noted in most of the nuclei, in Lhr background, and transcribed at higher rate than that of the single X chromosome of male. In hybrid females (both viable, e.g., C(mel)X(mel)X(sim); A(mel)A(sim) and rescued, e.g., C(mel)X(mel)X(mel); A(mel)A(sim)), the functional morphology of the X chromosomes were comparable to that of diploid autosomes in presence of one dose of Lhr. In hybrid metafemales (C(mel)X(mel)X(mel)X(sim); A(mel)A(sim)), two dose of melanogaster X chromosomes and one dose of simulans X chromosome were transcribed almost at 'female' rate in hybrid genetic background in presence of one dose of Lhr. In rescued hybrid males, the melanogaster-derived X chromosome appeared to complete its replication faster than autosomes. These results together have been interpreted to have suggested that Lhr suppresses the lethality of hybrids by regulating functional activities of the X chromosome(s) for dosage compensation.

Intrapleural streptokinase in complicated parapneumonic effusions and empyema.

BACKGROUND: The management of complicated parapneumonic effusions (CPE) and empyema by chest tube drainage usually fails because of thick viscous fluid and multiple pleural space loculations. The use of intrapleural fibrinolytic agents facilitates pleural drainage and can obviate the need for more invasive surgical interventions in these types of effusions. OBJECTIVE: To evaluate the role of intrapleural fibrinolytic therapy with streptokinase (STK) as an adjunctive therapy in the management of CPE and empyema. MATERIAL AND METHODS: Thirty patients of CPE and empyema were considered for intrapleural fibrinolytic therapy when the chest tubes/catheter drainage became insignificant (i.e., less than 50 ml a day) and the tube was adequately positioned and patent. Intrapleural STK was administered eight hourly in the dosage of 2,50,000 IU in 50 ml of saline. The end points were volume of fluid drained and radiological resolution. RRESULTS:There were 24 (80%) patients with CPE and six (20%) with empyema, with a mean age of 35 years. The median of STK doses used were three in 26 (87%) cases and two in four (13%) cases. There was significant drainage (mean +/- SD) 1094 ml +/- 116 and radiological resolution in 26 (87%) cases. The only complication observed was transient chest pain in one and fever in two patients. CCONCLUSION:Intrapleural fibrinolytic therapy with STK is a safe and effective adjunctive therapy in the management of CPE and empyema.

Transmission of filarial infection through blood transfusion.

Microfilariae can be transmitted by blood transfusion and they may be circulated in the recipient's blood but they do not develop into adult worms. Mortality associated with transfusion associated filarial infection is not documented but it may give rise to morbidity in transfusion recipients in terms of allergic reaction. The present study was carried out to investigate the association of post transfusion reactions and filarial infections in an endemic area. About 11,752 transfusion recipients were followed up and in 15 months period, 47 (0.4%) post transfusion reactions (PTR) were reported. Routine investigations for post transfusion reaction were carried out in all 47 patients and their respective blood donor. Moreover, blood culture, microfilaria detection by concentration technique, filarial antibody and antigen detection (both by ELISA) were done in all subjects. Out of 47 patients showing post transfusion reaction, 29 (61.7%) patients developed allergic reaction. Eighteen (38.3%) patients having allergic reaction did not have previous history of blood transfusion and 14 (29.8%) of them received transfusion from blood donors who was either positive for microfilaria, filarial antigen or antibody. Microfilaremia was demonstrated in 4 (8.5%) patients and 5 (10.6%) blood donors. Microfilaria was concurrently present in 2 patients and their respective donors. Filarial antibody was detected in 27 (56.5%) patients and 26 (55.3%) blood donors but microfilaria was detected in 3 (6.4%) and 4 (8.5%) subjects, respectively. Antigen detection test correlated with microfileraemic state of subjects. The result shows that transfusion associated filarial infection may be a probable cause for transfusion-associated morbidity in endemic areas. In 14 (29.8%) patients having allergic reactions, the probable cause was transfusion-associated filarial infection. Filarial antigen detection test was found to be more useful in detecting infections. Blood donors with active history of filarial infection should be deferred from donating blood. Filarial antigen detection test may be employed as screening test for blood donors, if possible.

Correlation between human papillomavirus DNA detection in maternal cervical smears and buccal swabs of infants.

Acquisition of human papillomavirus (HPV) infection in buccal mucosa by the infants at birth has been investigated. Presence of HPV DNA was evaluated in cervical smears of 30 pregnant women before delivery and in buccal swabs of the corresponding 31 infants (1 set of twins). HPV DNA was detected among the 40% of women, 16% of infants an the detection was concordant in 23 mother-infant pairs. HPV DNA was demonstrated in buccal mucosal cells of 41.6% of the infants born to HPV positive mothers. Maternal-infant transmission was highest for HPV 6/11. Assessment of the risk of developing HPV related oral lesions in children at later life owing to acquisition of HPV during perinatal period may help in determining a strategy to combat the disease.

Longitudinal study of human milk creamatocrit and weight gain in exclusively breastfed infants.

OBJECTIVE: To determine the creamatocrit values of breastmilk at different lactational periods during the first 6 months after delivery and its relation to infant growth. DESIGN: Prospective follow-up study. SETTING: Maternity and well baby clinics of a metropolitan teaching, a rural teaching and a suburban municipal hospital. SUBJECTS: 1322 exclusively breastfed infants and their mothers. There were 160 preterm, 281 term low birth weight (LBW) and 881 term appropriate for date (AFD) babies. METHODS: Creamatocrit of breastmilk and infant's body weight were recorded at 4,8,12,16,20 and 24 weeks after birth. RESULTS: Creamatocrit values of breastmilk in all mothers increased upto 16 post partum weeks and then declined. There were no significant differences in creamatocrit values of breastmilk of mothers of preterm, term LBW and term non-LBW infants. Preterm and term LBW infants showed a catch up in body weight with the term AFD group by 20 weeks post-partum. CONCLUSIONS: The breastmilk creamatocrit progressively rises upto the fourth month of lactation but is not influenced by gestation or intra uterine growth retardation. There is no relationship between breastmilk creamatocrit and infant growth. Exclusive breast feeding adequately supports growth of LBW infants in comparison to their normal peers during the first 6 months of life.

Optimization of test conditions for development of MTT as in vitro screen.

The quick and easy method of tetrazolium based colorimetric assay with MTT [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was used to test the viability of the adult parasites of a rodent filariid Acanthocheilonema viteae in vitro. The ideal conditions required for antifilarial screening were determined by correlating the MTT reduction ability of worms with their size and age in the vertebrate host, also the duration of incubation and temperature of the in vitro culture. It was observed that the worms collected from the host after 90 days of L3 (infective larvae) exposure were not suitable for in vitro screen as they could not reduce MTT to that extent as the worms of early infection. Healthy and full grown worms and also those incubated at 37 degrees C for 16 hr or more caused maximum MTT reduction. Thus, it is recommended to select healthy adult filariids of proper age and size (male > 3.5 cm; female > 7.0 cm). The incubation temperature of the in vitro culture system needs to be adjusted to 37 degrees C and parasites might be exposed to drugs upto 24 hr without much alteration in MTT reduction of untreated controls.

Detection of type specific human papillomavirus (HPV) DNA in cervical cancers of Indian women.

Commercial Vira Pap and Vira Type kits of Life Technologies Inc., U.S.A., were used to determine prevalence and type specific distribution of human papilloma viruses (HPV) in 18 biopsy samples of cervical carcinomas and 26 specimens of exfoliated cervical cells (swabs). The women were either attending out-patient's department of a suburban hospital or a cancer hospital in Calcutta, India. HPV DNA was detected in 9 biopsy specimens but not in any of the cervical swabs. Five of the 9 HPV positive biopsies had HPV DNA type 16/18. Neither HPV 6/11 nor HPV 31/33/35 DNAs were detected in any of these 9 specimens. Results indicated possible presence of HPV DNAs of types other than the above in at least 4 specimens.

Carboline antifilarials: effects on carbohydrate metabolising enzymes in Litomosoides carinii female.

Two antifilarial compounds, viz., 90/55 (7-oxo-1-phenyl-8, 14-dihydropyrido (3,4-b) imidazo (1,2-c) quinazolo (4,5-g) and 87/639 (6-Nitro-1-phenyl-9H-pyrido (3,4-b) indole at 0.5 and 2.0 micron concentrations substantially inhibited glucose uptake and increased lactate production by L. carinii during in vitro incubation for 2 hr. The treated parasites, showed increased activities of glycogen phosphorylase, phosphofructokinase and pyruvate kinase. Hexokinase and fumarate reductase activities level in the worms were significantly lowered. Therefore it appears that both the compounds kill adult L. carinii by interfering with its carbohydrate metabolism.

Evaluation of vanillic acid analogues as a new class of antifilarial agents.

A number of vanillic acid analogues (1-14) have been synthesised and evaluated against experimental filarial infections using cotton rats (Sigmodon hispidus) infected with Litomosoides carinii, a primary screening model, at a dose of 30 mg/kg, ip for 5 days. Of the 8 compounds tested, 4 (5,7, 11 and 12) exhibited high micro- and macro-filaricidal activity with sterilization of surviving female worms. Compounds 5, 7, 12 showed remarkable adulticidal action (> 80%). Sterilization of the female worms by compounds 11 and 12 was highly significant (80-100%).

Relative performance of Organon kit in comparison to Du Pont for confirmatory serological testing of HIV infection by western blot test in sera from blood donors.

A total of 32 specimens with different categories of reactivity by Du Pont Western Blot kit comprising of specimens showing full spectrum of HIV-I antigen specific bands, 19 specimens showing total absence of bands and four specimens showing non-specific bands (without any interpretative importance) were subjected to Western Blot testing by Organon test. Of the nine specimens showing full spectrum of bands by Du Pont the correlation with Organon kit was 100 per cent based on WHO criteria. Four specimens with non-specific indeterminate band pattern by Du Pont failed to show any band in Organon kit, indicating that latter to be more specific.